1. Field of the Invention
The present invention relates to a human lactoferrin gene isolated from breast tissue and to the protein product encoded therein. The present invention further relates to the promotor region of human lactoferrin gene and to methods for detecting and analyzing malignancies arising from tissues that normally secrete lactoferrin using a novel human lactoferrin cDNA gene sequence.
2. Background Information
Lactoferrin is a single polypeptide molecule (M, 76,000) with sites where two oligosaccharide chains can attach (B. F. Anderson et al., J. Mol. Biol. 209:711-734 (1989)). This protein shares significant homology with transferrin, however, its role in iron transport is limited since it binds iron 260 times stronger than transferrin (B. F. Anderson et al., (1989)). Two and possibly three isoforms of lactoferrin have been isolated using an affinity chromatography (P. Furnamski et al., J. Exp. Med. 170:415-429 (1989); A. Kijlstra et al., Current Eye Res., 8:581-588 (1989)). Lactoferrin has been shown to inhibit bacterial growth by chelating iron and directly attacking the cell wall (R. T. Ellison et al., Infect Immun., 56:2774-2781 (1988)), contribute to the anemia of chronic disease (Birgens. Scand. J. Haematol., 33:225-230 (1984)), improve intestinal absorption of iron in infants (Birgens., (1984)) inhibit myelopoiesis (H. E. Broxmeyer et al., Blood Cells 13:31-48 (1987)), and degrade mRNA (P. Furmanski et al., (1989); M. R. Das et al., Nature 262:802-805 (1976); P. Furmanski and Z. P. Li, Exp. Hematol 18:932-935 (1990). Large quantities of lactoferrin are found in breast milk (B. Lonnerdal et al., Nutrition Report Int., 13:125-134 (1976)), in estrogen-stimulated uterine epithelium (B. T. Pentecost and C. T. Teng, J. Biol. Chem. 262:10134-10139 (1987)), and in neutrophilic granulocytes (P. L. Masson et al., J. Exp. Med., 130:643-658 (1969)) with smaller amounts in tears, saliva, serum, and seminal fluid (D. Y. Mason and C. R. Taylor, J. Clin. Path., 31:316-327 (1978)).
While normal breast ductal epithelium and neutrophilic granulocytes contain lactoferrin, their malignant counterparts frequently do not (C. Charpin et al., Cancer, 55:2612-2617 (1985); T. A. Rado et al., Blood, 70:989-993 (1987)). This has been evaluated at the protein level and in a few samples at the messenger RNA level (T. A. Rado et al., (1987)). Analysis at the genomic level has not been performed. DNA variations, that are detected in the coding regions, may lead to abnormal protein structure and loss of normal function. Variations, such as mutations, deletions, or changes in methylation, at the promoter regions could lead to altered regulation of the gene. Evaluation of the lactoferrin gene may provide interesting insight concerning the production of lactoferrin in malignant cells. Thus, the need exists for the structure of the lactoferrin gene including the cDNA and the promotor region. The present invention provides such a description of the structure of a human lactoferrin cDNA and promotor region of the gene.
Using a lactoferrin cDNA clone isolated from human breast tissue, the applicants have evaluated restriction fragment length changes in DNA from the white blood cells of 10 normal controls, acute non-lymphocyte leukemia (ANLL) cells from 7 patients, T-cell acute lymphocyte leukemia (ALL) from one patient, 3 leukemia cell lines, and 7 breast cancer cell lines. A comparative study of the lactoferrin gene in these different cell types is provided herein.
The present invention further relates, in part, to a human lactoferrin cDNA and the protein product encoded therein. In another aspect, the present invention relates to methods for detecting malignancy in tissues that normally secrete lactoferrin by evaluating restriction patterns in DNA using a lactoferrin gene probe of the present invention.